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Abstract

Over the recent years, immunotherapeutic approaches, especially immune checkpoint blockade-based therapies, have been found to be very effective to treat specific cancer types. However, many cancer patients do not respond to these currently available immune-system stimulating therapies or become resistant. Previously, this has been linked to tumor infiltrating immunosuppressive myeloid cells such as tumor-associated macrophages of the M2-like polarized phenotype and myeloid-derived suppressor cells. In the search for new approaches to treat cancer, this thesis was therefore aiming to gain a better understanding of the biology of the myeloid cell compartment in the tumor microenvironment and find novel modifiers of macrophage polarization. This work presents a new methodology using CRISPR/Cas9-based screening to identify druggable targets promoting the immunosuppressive (M2) or preventing the proinflammatory (M1) macrophage phenotype. Inhibiting these targets will reinstruct tumor-infiltrating myeloid cells to stimulate the antitumor immune response in the tumor microenvironment. As genetic perturbation of primary monocytes is very challenging, human monocytic THP-1 cells were used as surrogates to study macrophage biology. A THP-1 cell clone was engineered to stably express Cas9 that enables the performance of CRISPR/Cas9-based functional genomics studies. Myeloid cell transcriptome and whole genome targeting pooled CRISPR/Cas9 screens were performed detecting the effects of sgRNA-mediated knockout on CD80 expression in differentiated and polarized THP-1 cells. Thereby, 170 genes potentially involved in M2 polarization were identified. Some of the hits, such as OGT and TNFAIP3 have been described before to have a role in macrophage biology and polarization, supporting the validity of the screening approach. To confirm the screening results an RNP/gRNA-based CRISPR/Cas9 validation approach was developed, which will allow further characterization of the identified targets both in THP-1 cells but most importantly in human primary monocytes/macrophages. As readouts, the effects of gene knockouts on the expression of M1 macrophage markers and on secretion of various proinflammatory cytokines and chemokines are measured. Investigating a first selection of 20 hits using this approach, the screening data of knockout of the transcriptional regulators GFI1 and OTX1, the histone deacetylase KDM1A, as well as TNFAIP3 could be verified. More extensive experimental work has been performed to better understand the function of TNFAIP3 in macrophage polarization. Knockout of TNFAIP3 in primary myeloid cells promoted the M1 polarization of the cells under both M1 and M2 polarizing conditions. Furthermore, in coculture-experiments it was shown that the knockout cells are able to activate T cells better than the control cells. To evaluate the role of the potentially druggable deubiquitinase function in macrophage polarization, Jurkat cells were genetically engineered to only express TNFAIP3 with a function-inhibiting C103A mutation in the de-ubiquitinase domain. RNA-seq experiments however implicate that the deubiquitinase function of TNFAIP3 does not seem to be essential for mediating the therapeutically relevant effect on macrophage polarization seen in TNFAIP3 knockout cells. Other options of targeting TNFAIP3 are now being investigated. Based on the results generated in this thesis, projects have been started to further study the role of the screening hits in macrophage polarization to evaluate their potential as starting points for a pharmaceutical development program. Also, an extended target identification project is planned in mouse models.